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Melks as modifiers of the rac pathway and methods of use

Melks as modifiers of the rac pathway and methods of use description/claims

This application claims priority to U.S. provisional patent application 60/495,193 filed Aug. 14, 2003. The contents of the prior application are hereby incorporated in their entirety.

Cell movement is an important part of normal developmental and physiological processes (e.g. epiboly, gastrulation and wound healing), and is also important in pathologies such as tumor progression and metastasis, angiogenesis, inflammation and atherosclerosis. The process of cell movement involves alterations of cell-cell and cell-matrix interactions in response to signals, as well as rearrangement of the actin and microtubule cytoskeletons. The small GTPases of the Rho/Rac family interact with a variety of molecules to regulate the processes of cell motility, cell-cell adhesion and cell-matrix adhesion. Cdc42 and Rac are implicated in the formation of filopodia and lamellipodia required for initiating cell movement, and Rho regulates stress fiber and focal adhesion formation. Rho/Rac proteins are effectors of cadherin/catenin-mediated cell-cell adhesion, and function downstream of integrins and growth factor receptors to regulate cytoskeletal changes important for cell adhesion and motility.

There are five members of the Rho/Rac family in the C. elegans genome. rho-1 encodes a protein most similar to human RhoA and RhoC, cdc-42 encodes an ortholog of human Cdc42, and ced-10, mig-2 and rac-2 encode Rac-related proteins. ced-10, mig-2 and rac-2 have partially redundant functions in the control of a number of cell and axonal migrations in the worm, as inactivation of two or all three of these genes causes enhanced migration defects when compared to the single mutants. Furthermore, ced-10; mig-2 double mutants have gross morphological and movement defects not seen in either single mutant, possibly as a secondary effect of defects in cell migration or movements during morphogenesis. These defects include a completely penetrant uncoordinated phenotype, as well as variably penetrant slow-growth, vulval, withered tail, and sterility defects, none of which are seen in either single mutant.

MELK (maternal embryonic leucine zipper kinase) is a member of the evolutionarily conserved KIN1/PAR-1/MARK kinase family which is involved in cell polarity and microtubule dynamics.

The ability to manipulate the genomes of model organisms such as C. elegans provides a powerful means to analyze biochemical processes that, due to significant evolutionary conservation, have direct relevance to more complex vertebrate organisms. Due to a high level of gene and pathway conservation, the strong similarity of cellular processes, and the functional conservation of genes between these model organisms and mammals, identification of the involvement of novel genes in particular pathways and their functions in such model organisms can directly contribute to the understanding of the correlative pathways and methods of modulating them in mammals (see, for example, Dulubova I, et al, J Neurochem 2001 April; 77(1):229-38; Cai T, et al., Diabetologia 2001 January; 44(1):81-8; Pasquinelli A E, et al., Nature. 2000 Nov. 2; 408(6808):37-8; Ivanov I P, et al., EMBO J 2000 Apr. 17; 19(8):1907-17; Vajo Z et al., Mamm Genome 1999 October; 10(10):1000-4). For example, a genetic screen can be carried out in an invertebrate model organism having underexpression (e.g. knockout) or overexpression of a gene (referred to as a “genetic entry point”) that yields a visible phenotype. Additional genes are mutated in a random or targeted manner. When a gene mutation changes the original phenotype caused by the mutation in the genetic entry point, the gene is identified as a “modifier” involved in the same or overlapping pathway as the genetic entry point. When the genetic entry point is an ortholog of a human gene implicated in a disease pathway, such as RAC, modifier genes can be identified that may be attractive candidate targets for novel therapeutics.

All references cited herein, including patents, patent applications, publications, and sequence information in referenced Genbank identifier numbers, are incorporated herein in their entireties.

We have discovered genes that modify the RAC pathway in C. elegans, and identified their human orthologs, hereinafter referred to as Maternal Embryonic Leucine Zipper Kinase (MELK). The invention provides methods for utilizing these RAC modifier genes and polypeptides to identify MELK-modulating agents that are candidate therapeutic agents that can be used in the treatment of disorders associated with defective or impaired RAC function and/or MELK function. Preferred MELK-modulating agents specifically bind to MELK polypeptides and restore RAC function. Other preferred MELK-modulating agents are nucleic acid modulators such as antisense oligomers and RNAi that repress MELK gene expression or product activity by, for example, binding to and inhibiting the respective nucleic acid (i.e. DNA or mRNA).

MELK modulating agents may be evaluated by any convenient in vitro or in vivo assay for molecular interaction with a MELK polypeptide or nucleic acid. In one embodiment, candidate MELK modulating agents are tested with an assay system comprising a MELK polypeptide or nucleic acid. Agents that produce a change in the activity of the assay system relative to controls are identified as candidate RAC modulating agents. The assay system may be cell-based or cell-free. MELK-modulating agents include MELK related proteins (e.g. dominant negative mutants, and biotherapeutics); MELK-specific antibodies; MELK-specific antisense oligomers and other nucleic acid modulators; and chemical agents that specifically bind to or interact with MELK or compete with MELK binding partner (e.g. by binding to a MELK binding partner). In one specific embodiment, a small molecule modulator is identified using a kinase assay. In specific embodiments, the screening assay system is selected from a binding assay, an apoptosis assay, a cell proliferation assay, an angiogenesis assay, and a hypoxic induction assay.

In another embodiment, candidate RAC pathway modulating agents are further tested using a second assay system that detects changes in the RAC pathway, such as angiogenic, apoptotic, or cell proliferation changes produced by the originally identified candidate agent or an agent derived from the original agent. The second assay system may use cultured cells or non-human animals. In specific embodiments, the secondary assay system uses non-human animals, including animals predetermined to have a disease or disorder implicating the RAC pathway, such as an angiogenic, apoptotic, or cell proliferation disorder (e.g. cancer).

The invention further provides methods for modulating the MELK function and/or the RAC pathway in a mammalian cell by contacting the mammalian cell with an agent that specifically binds a MELK polypeptide or nucleic acid. The agent may be a small molecule modulator, a nucleic acid modulator, or an antibody and may be administered to a mammalian animal predetermined to have a pathology associated with the RAC pathway.

A genetic screen was designed to identify modifiers of the Rac signaling pathway that also affect cell migrations in C. elegans, where various specific genes were silenced by RNA inhibition (RNAi) in a ced-10; mig-2 double mutant background. The 4B260 gene was identified as a modifier of the RAC pathway. Accordingly, vertebrate orthologs of this modifier, and preferably the human orthologs, MELK genes (i.e., nucleic acids and polypeptides) are attractive drug targets for the treatment of pathologies associated with a defective RAC signaling pathway, such as cancer.

In vitro and in vivo methods of assessing MELK function are provided herein. Modulation of the MELK or their respective binding partners is useful for understanding the association of the RAC pathway and its members in normal and disease conditions and for developing diagnostics and therapeutic modalities for RAC related pathologies. MELK-modulating agents that act by inhibiting or enhancing MELK expression, directly or indirectly, for example, by affecting a MELK function such as enzymatic (e.g., catalytic) or binding activity, can be identified using methods provided herein. MELK modulating agents are useful in diagnosis, therapy and pharmaceutical development.

Nucleic Acids and Polypeptides of the Invention

Sequences related to MELK nucleic acids and polypeptides that can be used in the invention are disclosed in Genbank (referenced by Genbank identifier (GI) number) as GI#s 7661973 (SEQ ID NO: 1), 15559348 (SEQ ID NO:2), and 33878460 (SEQ ID NO:5) for nucleic acid, and GI# 7661974 (SEQ ID NO:6) for polypeptide. Additionally, nucleic acid sequences of SEQ ID NO:3 and SEQ ID NO:4 can also be used in the invention.

The term “MELK polypeptide” refers to a full-length MELK protein or a functionally active fragment or derivative thereof. A “functionally active” MELK fragment or derivative exhibits one or more functional activities associated with a full-length, wild-type MELK protein, such as antigenic or immunogenic activity, enzymatic activity, ability to bind natural cellular substrates, etc. The functional activity of MELK proteins, derivatives and fragments can be assayed by various methods known to one skilled in the art (Current Protocols in Protein Science (1998) Coligan et al., eds., John Wiley & Sons, Inc., Somerset, N.J.) and as further discussed below. In one embodiment, a functionally active MELK polypeptide is a MELK derivative capable of rescuing defective endogenous MELK activity, such as in cell based or animal assays; the rescuing derivative may be from the same or a different species. For purposes herein, functionally active fragments also include those fragments that comprise one or more structural domains of a MELK, such as a kinase domain or a binding domain. Protein domains can be identified using the PFAM program (Bateman A., et al., Nucleic Acids Res, 1999, 27:260-2). For example, the kinase domain (PFAM 00069) of MELK from GI# 7661974 (SEQ ID NO:6) is located at approximately amino acid residues 11-263. Further, the kinase associated domain 1 (PF02149) of the same protein is located approximately at amino acid residues 602 to 651. Methods for obtaining MELK polypeptides are also further described below. In some embodiments, preferred fragments are functionally active, domain-containing fragments comprising at least 25 contiguous amino acids, preferably at least 50, more preferably 75, and most preferably at least 100 contiguous amino acids of a MELK. In further preferred embodiments, the fragment comprises the entire functionally active domain.

• Provisional Patent
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